[41] Identification of atrial natriuretic peptide receptors in cultured cells

436

GUANYLYLCYCLASE

[41]

[41] I d e n t i f i c a t i o n o f A t r i a l N a t r i u r e t i c P e p t i d e R e c e p t o r s Cultured Cells B y DALE C.

in

LEITMAN, SCOTT A. WALDMAN,and FERID MURAD

Introduction Atrial natriuretic peptide (ANP) is a 28 amino acid peptide that was initially isolated from the atria of the heart.l Recently, it has become clear that ANP is a m e m b e r of a distinct class ofpeptides that are distinguished by the presence of a 17 amino acid disulfide ring structure and by their capacity to elicit a spectrum of physiological effects that include hypotension, natriuresis, and diuresis. 2 Presently, the natriuretic peptide family includes ANP, the 26 amino acid brain natriuretic peptide (BNP),3 and a recently discovered novel atrial peptide. 4 Animal and human studies indicate that ANP is an important regulator of blood pressure and vascular volume during physiological and pathological states.5 The development of drugs that mimic A N P to treat renal and cardiovascular disorders is a major goal that requires an understanding of the mechanism of action of ANP. The physiological effects of A N P are mediated by A N P receptors. Scatchard analysis of radioligand binding studies initially indicated that there was only a single class o f A N P receptors. However, two functionally and physically distinct classes of A N P receptors were originally identified by cyclic G M P (cGMP) response and cross-linking studies.6-8 The presence of two A N P receptor subtypes was definitively demonstrated by the purification 9-H and molecular cloning of the c D N A for both receptor subT. G. Flynn, M. L. de Bold, and A. J. de Bold, Biochem. Biophys. Res. Commun. 117, 859 (1983). 2 A. de Bold,

Science 230, 767 (1985).

3 T. Sudoh, K. Kangawa, N. Minamino, and H. Matsuo, Nature (London) 332, 78 (1988). 4 T. G. Flynn, A. Brar, L. Tremblay, I. Sarda, C. Lyons, and D. B. Jennings, Biochem. Biophys. Res. Commun. 161, 830 (1989). 5 S. A. Atlas, Recent Prog. Horm. Res. 42, 207 (1986). 6 D. C. Leitman and F. Murad, Biochim. Biophys. Acta 885, 74 (1986). 7 D. C. Leitman, J. W. Andresen, T. Kuno, Y. Kamasaki, J.-K. Chang, and F. Murad, J. Biol. Chem. 261, 11650(1986). s D. C. Leitman, C. R. Molina, S. A. Waldman, and F. Murad, UCLA Symp. Mol. Cell. Biol. 81, 39 (1988). 9 T. Kuno, J. W. Andresen, Y. Kamisaki, S. A. Waldman, L. Y. Chang, S. Saheki, D. C. Leitman, M. Nakane, and F. Murad, J. Biol. Chem. 261, 5817 (1986).

METHODS IN ENZYMOLOGY, VOL. 195

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

[41]

IDENTIFICATION OF ANP RECEPTORS

437

t y p e s . 12'13 The ANP-R1 receptor is a 130-kDa glycoprotein that contains

two functional domains joined by a single transmembrane region. The extracellular domain functions as an ANP receptor, whereas the intracellular domain contains guanylyl cyclase enzymatic activity that converts GTP to cGMP. The precise mechanism whereby the binding of ANP activates the guanylyl cyclase domain is unknown. However, recent studies suggest that accessory proteins may be regulated by ANP, which then may participate in the activation of guanylyl cyclase, 14Binding of ANP to the ANPR1 receptor results in the formation of cGMP, which is the second messenger that mediates most of physiological effects of ANP, such as smooth muscle relaxation, natriuresis, and diuresis. 15 The second ANP receptor, designated ANP-R2, may exist in the membrane in two forms, a 130-kDa dimer composed of two identical 66-kDa subunits joined by disulfide bonds and a 66-kDa monomer, l°,16 Despite the purification l° and cloning 12 of the cDNA for this receptor subtype, the physiological role of this receptor has remained a mystery. This receptor subtype is devoid of intrinsic guanylyl cyclase activity I° and apparently is not functionally coupled to the reported inhibition of adenylyl cyclase activity.17 It has been shown that the ANP-R2 receptor is associated with the activation of phospholipase C and the increased formation of the inositol phosphates and diacylglycerol) 8 However, no physiological effects of ANP have been shown to be mediated by inositol phosphates or diacylglycerol. It has been proposed by Maack and coworkers that the ANP-R2 receptor functions as clearance receptor that binds, internalizes, and degrades ANP. 19 The physiological basis for a

l0 D.Shenk, M. N. Phelps, J. G. Porter, R. M. Scarborough, G. A. McEnroe, and J. A. Lewicki, Proc. Natl. Acad. Sci. U.S.A. 84, 1521 (1987). II R. Takayanagi, L. M. Snajdar. T. Imada, M. Tamura, K. N. Pandey, K. S. Misono, and T. Inagami, Biochem. Biophys. Res. Commun. 144, 244 (1987). 12 F. Fuller, J. G. Porter, A. E. Arfsen, J. Miller, J. W. Schilling, R. M. Scarborough, J. A. Lewicki, and D. B. Shenk, J. Biol. Chem. 19, 9395 (1988). 13 M. Chinkers, D. L. Garbes, M.-S. Chang, D. G. Lowe, H. Chin, D. V. Goeddel, and S. Schulz, Nature (London) 338, 78 (1989). 14 C.-H. Chang, K. P. Kohse, B. Chang, M. Hirata, and F. Murad, FASEB J. 3, AI005 (1989). 15 D. C. Leitman and F. Murad, Endocrinol. Metab. Clin. North Am. 16, 79 (1987). 16 D. C. Leitman, J. W. Andresen, R. M. Catalano, S. A. Waldman, J. J. Tuan, and F. Murad, J. Biol. Chem. 263, 3720 (1988). z7 M. B. Anand-Srivastava, D. J. Franks, M. Cantin, and J. Genest, Biochem. Biophys. Res. Commun. 121, 855 (1984). ~8M. Hirata, C.-H. Chang, and F. Murad, Biochim. Biophys. Acta 1010, 346 (1989). 19T. Maack, M. Suzuki, F. A. Almeida, D. Nussenzveig, R. M. Scarborough, G. A. McEnroe, and J. A. Lewicki, Science 238, 675 (1987).

438

GUANYLYLCYCLASE

[41]

receptor that functions exclusively as a clearance receptor for ANP is unclear. ANP receptors are present in a variety of different cell types, including cells that are not involved in volume and blood pressure regulation, suggesting that ANP exerts other actions in addition to its well-characterized renal and cardiovascular effects. The properties and the regulation of the ANP receptors can be characterized by radioligand binding studies. Furthermore, the two ANP receptor subtypes can be distinguished by using selective atrial peptide analogs in cross-linking, competition binding, and cGMP response studies. In this chapter, we describe our methods for characterizing ANP receptors and the cGMP response to ANP in cultured cells 6,7,16,20,21 Iodination of ANP The first step in the development of a receptor binding assay is to prepare a radioactive ligand. Most ANP binding studies have used iodinated forms of the 26 amino acid peptide ANP(101-126) or the 28 amino acid peptide ANP(99-126). Both of these peptides contain a single tyro sine residue that is located at the carboxyl terminus and which can be iodinated. In our binding studies, we use the 26 amino acid atrial peptide that is iodinated by the Iodogen method. 22 Prior to iodination, microfuge tubes are coated with Iodogen. One milligram of Iodogen is dissolved in 25 ml dichloromethane, and then 50 ~l is added to the bottom of a microfuge tube. The Iodogen is then completely evaporated by a gentle stream of nitrogen gas. Once the tube is free of liquid it is used for the iodination of ANP. For the iodination reaction, I0 ~l of 50 mM sodium phosphate, pH 7.4, is added to a microfuge tube coated with Iodogen. Five microliters of ANP (0.2 nmol) dissolved in 10 mM acetic acid is then added. After the addition of I mCi Na1251, the reaction is incubated for 10 rain at room temperature. The unbound iodide is separated from the labeled peptide with a SepPak C18 column. During the iodination reaction a Sep-Pak C18 column is prepared by washing the column with 5 ml of 0. I% trifluoroacetic acid in I00% acetonitrile, followed by I0 ml of 0. I% trifluoroacetic acid in water. After the iodination reaction, the sample is removed from the microfuge 20 D. C. Leitman, V. L. Agnost, J. J. Tuan, J. W. Andresen, and F. Murad, Biochem. J. ?.44, 69 (1987). 21 D. C. Leitman, V. L. Agnost, R. M. Catalano, H. Schroder, S. A. Waldman, B. M. Bennett, J. J. Tuan, and F. Murad, Endocrinology 122, 1478 (1988). 22 p. R. P. Salacinski, C. McLean, J. E. C. Sykes, V. V. Clement-Jones, and P. J. Lowry, Anal. Biochem. 117, 136 (1981).

[41]

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tube and put at the bottom of a plastic syringe containing the Sep-Pak C~s column. The microfuge tube is washed with 500/zl 0.1% trifluoroacetic acid in water, which is then added to the syringe. The free iodide is then slowly eluted. The column is subsequently washed with 20 ml of 0.1% trifluoroacetic acid in water to remove free iodide. The 12SI-labeled ANP is eluted into a plastic centrifuge tube with 3 ml 0.1% trifluoroacetic acid in 100% acetonitrile. The sample is concentrated to approximately 1 ml under a continuous stream of nitrogen gas and then stored at 4°. The specific radioactivity of ANP ranges from 700 to 1400 Ci/mmol. Recently, iodinated ANP that is purified by high-performance liquid chromatography (HPLC) has become commercially available. However, we have obtained similar results when comparing ANP that we labeled to purified ANP purchased from companies.

ANP Receptor Assay We have used the following binding assay to characterize the ANP receptors in a variety of cultured cell types, including aortic endothelial and smooth muscle cells, fibroblasts, and Leydig cells. 6,7,16,2°We perform binding studies at 37° in order to compare the properties of the ANP receptors under the same conditions we used to determine the biological response (i.e., cGMP determinations) of ANP. For binding assays, 25,000 cells are plated into 24-well tissue culture dishes.The cells are then grown until confluency. The cells are washed twice with 1 ml serum-free Dulbecco's modified Eagle's medium (DMEM), pH 7.3, containing 3.7 g/liter sodium bicarbonate. The binding assay is initiated by the addition of 200 /A of DMEM, pH 7.4, containing 25 mM HEPES, 2 mg/ml bovine serum albumin, and lasI-labeled ANP. The cells are incubated in a 5% CO2 incubator maintained at 37° for 15-30 min. The unbound ANP is removed by rapidly washing the cells 4 times with ice-cold Hanks' balanced salt solution (HBSS) containing 2 mg/ml bovine serum albumin. After the cells are washed, they arc solubilized by addition of 500 ~I of 1 N sodium hydroxide. The cell lysate is removed after at least 1 hr of incubation at room temperature. An additional 500 ~1 of 1 N sodium hydroxide is used to wash the wells. The two samples are pooled, and the radioactivity is determined with a gamma counter. To determine nonspecific binding, parallel cultures are exposed to the same concentration of ~25I-labeledANP in the presence of 100 nM unlabeled ANP(101-126). Nonspecific binding of ANP to cultured cells usually does not exceed 10% of total binding. Specific binding is calculated by subtracting nonspecific binding from total binding.

440

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FIG. 1. Time course of specific binding of 125I-labeledANP to confluent bovine aortic endothelial cells. Endothelial cells were incubated with 150 pM 125I-labeledANP for various times at 37° in the absence and presence of 100 nM unlabeled ANP(8-33). Nonspecific binding was less than 2% of total binding.

ANP Binding Characteristics At 37 ° the binding of ANP to cells is rapid, and equilibrium is reached in about 15 min (Fig. 1). In most cell types, we find that ANP binding is stable for at least 30 min after equilibrium is reached. However, in rat lung fibroblasts maximal ANP binding is maintained until 40 min, after which binding drops sharply. 2° By 60 min specific ANP binding is only 50% of the binding observed at 30 min, probably due to degradation of ANP. These results indicate that ANP binding studies should be restricted to 15-30 min at 37 °. The binding of ANP to intact cultured cells displays a typical saturation isotherm. Figure 2 shows the effect of adding increasing concentrations of ANP on total, specific, and nonspecific binding in bovine aortic endothelial cells. 6 Specific ANP binding becomes saturated at approximately 0.5 nM. In contrast, nonspecific binding increases in a linear fashion. Scatchard analysis of the specific ANP binding data results in a straight line, suggesting that these cells contain a single class of binding sites. We later discovered that there are actually two ANP receptors present in cells, but the affinity of the two sites are nearly identical, accounting for the linear Scatchard plot. 7 The binding of ANP to cultured

[41]

IDENTIFICATIONOF ANP RECEPTORS I

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cells is of high affinity, with a Ko value in the range of 0.1-2 nM.16 When performing saturation curves, we typically use a range of 0.005-3 nM 1251l a b e l e d ANP. Whereas the K a values are very similar in different cell types, the number of ANP receptors varies substantially (Table I). The richest source of ANP receptors are aortic smooth muscle cells, with approximately 300,000 receptors/cell) 6 Another feature of ANP receptors is that the binding of ANP can be inhibited by a variety of ANP analogs. Analogs lacking the amino-terminal amino acids are nearly as effective at competing for ANP binding sites as the native ANP. Atrial peptides with a deletion of the carboxy-terminal amino acids phenylalanine-arginine-tyrosine 6 and ANP analogs with amino acid substitutions 23 effectively compete for ANP receptors, despite their having little or no intrinsic biological activity in some cell types. 23 R. M. Scarborough, D. Shenk, G. A. McEnroe, A. E. Arfsen, L.-L. Kang, K. Schwartz, and J. A. Lewicki, J. Biol. Chem. 261, 12960 (1986).

442

GUANYLYL CYCLASE

[41]

TABLE I A N P RECEPTOR BINDING AND EFFECTS OF A N P ON c G M P AND PARTICULATE GUANYLYL CYCLASE ACTIVITY IN CULTURED CELLS a

Stimulation (-fold) Cell type

cGMP

Guanylyl cyclase

Receptors/cell

Ko (nM)

BAC HLF MDCK BASM RME RL MDBK BAE

13 35 58 60 120 260 300 475

1.5 3.1 3.2 2.5 5.0 7.0 7.8 8.0

50,000 80,000 N.D. 310,000 N.D. 3000 N.D. 14,000

0.12 0.32 0.82 0.11 0.09

a The values for ANP stimulation ofcGMP accumulation and paniculate guanylyl cyclase were obtained from the concentration-response curve using an ANP concentration of 100 nM and 1 /zM, respectively. The ANP receptor number and affinity were obtained from the saturation curves. The cell types shown are bovine aortic endothelial (BAE), bovine adrenal cortical (BAC), bovine aortic smooth muscle (BASM), human lung fibroblasts (HLF), rat Leydig (RL), rat mammary epithelial (RME), Madin-Darby canine kidney (MDCK), and Madin-Darby bovine kidney (MDBK) cells. N.D., None detectable (i.e., <3000 sites/cell). A N P Cross-Linking Cross-linking studies are useful in characterizing the molecular properties of A N P receptors. Specifically, cross-linking of A N P to intact cells has b e e n instrumental for the elucidation of the molecular size and subunit composition of A N P receptors and the identification of multiple A N P r e c e p t o r subtypes. A N P has been successfully cross-linked to receptors by either chemical affinity or photoaffinity cross-linking techniques. Both methods h a v e yielded similar results. We have used chemical cross-linking techniques since no special equipment is necessary and the same ligand can be used for binding and cross-linking studies. The cross-linking reagent m o s t c o m m o n l y used to cross-link A N P to its receptor is disuccinimidyl suberate (DSS). Once A N P is covalently bound to its receptors by DSS, the molecular size of ANP-binding proteins can be determined by comparing the mobility of the A N P - r e c e p t o r complex with k n o w n protein standards on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Cultured cells are plated at a density of 50,000 cells in 6-well tissue culture plates (35 mm). W h e n the cells reach confluence they are washed

[41]

IDENTIFICATION OF ANP RECEPTORS

443

3 times with HBSS containing 10 mM HEPES, pH 7.3. The cells are incubated for 1 hr at room temperature with 1 ml HBSS containing 1-2 nM 125I-labeled ANP in the absence and presence of unlabeled ANP. Just before the end of the 1-hr incubation time point, DSS is dissolved in dimethyl sulfoxide to a final concentration of 50 mM. The 50 mM DSS stock solution is diluted into HBSS (4/~l/ml) to a final concentration of 0.2 mM. One milliliter of HBSS containing DSS is added to the cells, which are then incubated for an additional 30 min at room temperature. The medium is aspirated, and the cells are washed quickly with 2 ml of 50 mM Tris-HC1, pH 7.3. Four hundred microliters of SDS sample buffer (62.5 m M Tris-HCl, pH 7.6, 10% glycerol, and 2.3% SDS) is added to the cells, and the plates are placed on top of a thin layer of boiling water for 3 min. The solubilized cells are removed and split into two equal portions. 2-Mercaptoethanol is added to one portion to a final concentration of 5%. The samples are electrophoresed on a 7.5% SDS-polyacrylamide gel. The gel is dried on Whatman 3 MM paper and exposed to Kodak Omat X-ray film at - 70 °. Figure 3 shows the autoradiograms of ANP that were cross-linked to intact cultured cells. ANP cross-links to 66- and 130-kDa proteins under both nonreducing and reducing conditions.16 However, the proportion of these two bands varies markedly in the presence of reducing agents such as 2-mercaptoethanol. Under nonreducing conditions approximately 40% of the ANP binding sites have a molecular mass of 130 kDa, whereas the remaining binding sites have a molecular mass of 66 kDa. In contrast, when the proteins are reduced prior to electrophoresis, over 95% of the ANP binding sites consist of the 66-kDa protein. These results suggest that ANP receptors exist in three molecular forms in intact cells: (1) a nonreducible 130-kDa protein that consists of a single polypeptide, (2) a 130-kDa dimer protein that is made up of two identical 66-kDa subunits bridged by disulfide bonds, and (3) a 66-kDa protein that exists as a monomer. We previously designated the 130-kDa nonreducible protein as ANP-RI and the 130-kDa dimer and the 66-kDa monomer ANP-binding protein as ANP-R2. 7 These two ANP receptors have been purified, 9-~1and the cDNA for each receptor subtype has been isolated. ]2:3 In addition to the different subunit structure and amino acid composition, the two ANP receptors have different pharmacological and functional properties. The ANP-R1 receptor has intrinsic guanylyl cyclase activity and has been shown to mediate many of the physiological responses of ANP. The ANP-R2 receptor is devoid of guanylyl cyclase activity 1° and is not associated with any known physiological effects of ANP. The ANPR1 receptor has much more rigid requirements for binding of ANP analogs compared to the ANP-R2 receptor. ANP analogs that are truncated at the

444

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FIG. 3. Autoradiograms of SDS-polyacrylamide gels of cultured cells after binding with 12SI-labeled ANP and cross-linking with DSS. Confluent cultures were incubated with 2 n M 125I-labeled ANP for 1 hr at room temperature in the absence ( - ) or presence ( + ) of 1 p.M ANP. The cells were then cross-linked by adding DSS to a final concentration of 0.1 mM for 30 min. The cells were solubilized with SDS sample buffer and subjected to SDS-PAGE under nonreducing (A) or reducing (B) conditions. The mobilities of molecular weight standards are given at left. The cell types shown are bovine aortic endothelial (BAE), bovine adrenal cortical (BAC), bovine aortic smooth muscle (BASM), human lung fibroblasts (HLF), rat Leydig (RL), rat mammary epithelial (RME), Madin-Darby canine kidney (MDCK), and Madin-Darby bovine kidney (MDBK) cells.

carboxy terminus and linear peptides lacking the disulfide bridge bind much more effectively to the ANP-R2 receptor compared with the ANPR1 receptor. Several ANP-R2 selective peptides, such as atriopeptin I, are commercially available. These peptides completely inhibit ANP binding to many cells in competition binding studies, but they do not antagonize the ANP-induced stimulation of cGMp.6,23'24 Unfortunately, selective agonists or antagonists for the guanylyl cyclase-ANP-R1 receptor are presently not available. 24 R. M. Scarborough, G. A. McEnroe, A. E. Affsen, L.-L. Kang, K. Schwartz, and J. A. Lewicki, J. Biol. Chem. 263, 16818 (1988).

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Cyclic G M P Determination We have shown previously that there are two functionally distinct ANP receptor subtypes in cultured cells. 6 Only the nonreducible ANP-R1 receptor mediates the ANP activation of guanylyl cyclase and increased cGMP levels. 7 Table I illustrates that certain cell types which have either no detectable or a small number of ANP receptors can exhibit a much greater biological response to ANP compared with cells with the greatest number of receptors. These results demonstrate that the number of ANP receptors does not reflect the biological response to ANP. These results suggest that the magnitude of the cGMP response may depend only on the number of ANP-R1 receptors or possibly the ratio of the ANP-R1 receptors to ANP-R2 receptors, rather than the total amount of ANP receptors. We believe that, in addition to performing competition binding studies, it is necessary to determine the stimulation of cGMP levels to evaluate the biological potency of various atrial peptide analogs. Measuring the cGMP response to ANP is also vital when comparing the number of ANP receptors after physiological perturbation and hormone or drug treatment. In different studies, the stimulation of cGMP has decreased, increased, or remain unaltered after the number of ANP receptors declined by ANP or angiotensin II treatment. 25-27These studies suggest that the two receptor subtypes can be independently regulated. Culture cells are plated in 6-well dishes at a density of 50,000 cells/35 mm dish. At confluence the cells are washed 2 times with 2 ml serum-free DMEM. The cells are then preincubated for 15 min in 990/xl DMEM, pH 7.3, containing 10 mM H E PE S and 0.5 mM 3-isobutyl-l-methylxanthine. After preincubation, l0/xl of ANP is added to the medium, and the cells are incubated for 3 or 5 min. To minimize ANP binding to the tubes, we use polystyrene tubes and dilute ANP in water that contains a carrier such as 0.01% bacitracin or ! mg/ml bovine serum albumin. The medium is aspirated, and 1 ml of 6% trichloroacetic acid is added to cells. The cell culture plates are stored at - 7 0 °. The plates are thawed at room temperature, and the cellular lysate is placed in 12 x 75 mm disposable glass test tubes. After centrifugation at 4000 rpm for 20 min at 4°, 850 ~1 of the supernatant fraction is placed in 13 x 100 mm disposable glass test tubes. Fifty microliters of 1 N HCI is added to each sample, and the 25y. Hirata, S. Hirose, S. Takagi, H. Matsubara, and T. Omae,Eur. J. Pharmacol. 135,439 (1987). 26p. Roubert, M.-O. Lonchampt,P. Chabrier, P. Plas, J. Goulin,and P. Braquet, Biochem. Biophys. Res. Commun. 26, 61 (1987). 27p. Chabrier, P. Roubert, M.-O. Lonchampt, P. Plas, and P. Braquet,J. Biol. Chem. 26, 13199 (1988).

446

GUANYLYLCYCLASE

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samples are extracted 5 times with 2 ml of water-saturated ether. The residual ether is evaporated by incubating the samples in a 55° water bath for 30 min. The samples are neutralized by the addition of 50/zl of I N sodium hydroxide and 50/zl of 1 M sodium acetate, pH 4.0. To acetylate the samples, 28 500/zl of the sample is removed to 12 × 75 mm test tubes. Twenty microliters triethylamine is added, and the tube is vortexed. Then 10 ~1 acetic anhydride is immediately added, and the tube is again vortexed. The cGMP content in the sample is measured by radioimmunoassay (see [30], this volume). ANP Stimulation of Cyclic GMP In the presence of the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine, ANP produces a maximal increase in intracellular cGMP content in 3-I0 min at 37o.6'16 The magnitude of the increase in cGMP levels and the concentration-response curves vary markedly in different cell types. Table I shows that ANP increases cGMP from 15- to 500-fold in various cell types. The most sensitive cell type that we have studied is bovine aortic endothelial cells. An approximate 5-fold increase in cGMP levels can be detected at l pM. The exquisite sensitivity of the BAE cells to ANP has permitted us to develop a bioassay for ANP in human plasma. The concentration-response curves for vascular smooth muscle, fibroblasts, and kidney cells are shifted to the right by I0- to 100-fold compared to BAE cells. 16The ECs0 for cGMP formation ranges from 100 pM in BAE cells to greater than I nM in some cell types, such as fibroblasts and vascular smooth muscle cells.

28j. F. Harper and G. Brooker,J. Cyclic Nucleotide Res. 1, 207 (1975).